Method of treating pain using an antagonist of GM-CSF

ABSTRACT

The present invention relates generally to a method for the treatment and prophylaxis of pain. In accordance with the present invention, it is proposed that antagonists of GM-CSF are effective in the treatment of pain. Antagonists of GM-CSF include, but are not limited to, antibodies which are specific for GM-CSF or the GM-CSF receptor. The present invention further provides transgenic animals, such as a GM-CSF knock-out mouse, useful for testing antagonists in certain disease models.

This application claims the benefit of U.S. Provisional Application No.61/139,687, filed Dec. 22, 2008, and U.S. Provisional Application No.61/164,491, filed Mar. 30, 2009, which are both incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to a method for the treatmentand prophylaxis of pain. In accordance with the present invention, it isproposed that antagonists of GM-CSF are effective in the treatment ofpain. Antagonists of GM-CSF include, but are not limited to, antibodieswhich are specific for GM-CSF or the GM-CSF receptor. The presentinvention further provides transgenic animals, such as a GM-CSFknock-out mouse, useful for testing antagonists in certain diseasemodels.

BACKGROUND OF THE INVENTION

Pain

Pain of any type is the most frequent reason for physician consultationin the United States, prompting half of all Americans to seek medicalcare annually. It is a major symptom in many medical conditions,significantly interfering with a person's quality of life and generalfunctioning. Diagnosis is based on characterizing pain in various ways,according to duration, intensity, type (dull, burning or stabbing),source, or location in body. Usually pain stops without treatment orresponds to simple measures such as resting or taking an analgesic, andit is then called acute pain. But it may also become intractable anddevelop into a condition called chronic pain, in which pain is no longerconsidered a symptom but an illness by itself.

Pain can be classified according to many schemes and circumstances.There are two basic types of pain: acute and chronic. Acute pain occursfor brief periods of time and is associated with temporary disorders.However, it is always an alarm signal that something may be wrong.Chronic pain is continuous and recurrent. It is associated with chronicdiseases and is one of their symptoms. Pain intensity not only dependson the type of stimulus that caused it, but also on the subjectiveperception of the pain. Despite a wide range of subjective perception,several types of pain have been classified according to:

-   -   The stimulus that caused the pain.    -   The pain's duration.    -   The features of pain (intensity, location, etc.).

Another classification system is as follows:

-   -   Gnawing pain. Continuous with constant intensity. It generally        worsens with movement.    -   Throbbing pain. This is typical of migraine pain. It is caused        by dilation and constriction of the cerebral blood vessels.    -   Stabbing pain. Intense and severe. It is caused by mechanical        stimuli.    -   Burning pain. A constant, burning feeling, like, for example,        the type of pain caused by heartburn.    -   Pressing pain. Caused by constriction of the blood vessels or        muscles.

There are also specific types of pain:

-   -   Muscle pain. Also known as myalgia, this pain involves the        muscles and occurs after excessive exertion or during        inflammation.    -   Colicky pain. Caused by muscle contractions of certain organs,        such as the uterus during the menstrual period. Generally cyclic        in nature.    -   Referred pain. Occurs when the painful sensation is felt in a        site other than the one where it is actually occurring,        depending upon how the brain interprets information it receives        from the body.    -   Post-surgical or Post-operative pain. Occurs after surgery and        is due to lesions from surgical procedures.    -   Bone cancer pain. Certain types of cancers, such as prostate,        breast, or other soft-tissue tumors, may progress to a painful        disorder of the bone known as metastatic bone disease.        Standard Care for Pain Treatment

There are many ways to treat pain. Treatment varies depending on thecause of pain. The main treatment options are as follows:

Acetaminophen: Tylenol (Acetaminophen) is used to treat pain. Unlikeseveral other medications for pain, Tylenol does not haveanti-inflammatory effects. Often, however, in cases of chronic pain, noinflammation is at the site of the pain, and thus Tylenol may be anappropriate treatment choice. Tylenol is safe when used appropriately,but can be dangerous when used excessively. Also, Tylenol may causeunwanted effects when used with certain other medicaments.

Non-Steroidal Anti-Inflammatory Medications (NSAIDs): The NSAIDs (suchas Ibuprofen, Motrin, Aleve, etc.) are most beneficial in cases of acutepain, or flare-ups in patients with chronic pain. NSAIDs are alsoexcellent at treating inflammatory conditions including tendonitis,bursitis, and arthritis. In general, NSAID use is limited for patientswith chronic pain because of concerns about the development to stomachproblems. While the newer, so-called COX-2 inhibitors, such as Celebrex,were designed to avoid this complication, caution should still be usedwhen using these medications for long periods of time.

Corticosteroids: As with NSAIDs, corticosteroids are powerfulanti-inflammatory medications, and best used for acute pain or forflare-ups of a chronic inflammatory problem. Corticosteroids can eitherbe taken orally (such as Medrol. Prednisone), or injected into the softtissues or joints (cortisone injections).

Narcotics: Narcotics should be considered if pain cannot be otherwisecontrolled. Many narcotics can be dangerous and addicting. Whilenarcotic medications are useful for acute pain, they also havesignificant side effects. The short-acting types of these medicationscan lead to overuse and the development of tolerance. Long-actingoptions have fewer side effects, and better control of chronic pain.Narcotics can become addictive when they are used for lengthy timeswithout gradual reduction in the dose, or if the medications are takenfor reasons other than pain.

Anti-Convulsants: Anti-convulsant medications are the category ofmedications that work to relieve nerve pain. These medications alter thefunction of the nerve and the signals that are sent to the brain. Themost commonly prescribed anticonvulsant medication for nerve pain iscalled Neurontin (Gabapentin). Another option that has more recentlyemerged, specifically for the treatment of fibromyalgia, is calledLyrica (Pregabalin).

Local Anesthetics: Local anesthetics can provide temporary pain reliefto an area. When used in the setting of chronic pain, local anestheticsare often applied as a topical patch to the area of pain. Lidoderm comesin a patch that is applied to the skin and decreases the sensitivity ofthis area.

All of the above mentioned treatment options have drawbacks, sideeffects, or use is limited to certain types of pain. Hence, there isstill a high unmet medical need for the treatment of pain.

GM-CSF

Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokinethat functions as a white blood cell growth factor. GM-CSF stimulatesstem cells to produce granulocytes (neutrophils, eosinophils, andbasophils) and monocytes. Monocytes exit the circulation and migrateinto tissue, whereupon they mature into macrophages. It is, thus, partof the natural immune/inflammatory cascade, by which activation of asmall number of macrophages can rapidly lead to an increase in theirnumbers, a process crucial for fighting infection. The active form ofGM-CSF is found extracellularly as a homodimer. In particular, GM-CSFhas been identified as an inflammatory mediator in autoimmune disorders,like rheumatoid arthritis (RA), leading to an increased production ofpro-inflammatory cytokines, chemokines and proteases and, thereby,ultimately to articular destruction.

GM-CSF is a cytokine which is involved in various processes in the humanand the animal body. Also, certain diseases and pathologies, such asinflammatory diseases, were recently linked to GM-CSF, and GM-CSF wassuggested as a potential point of intervention. The present inventiondiscloses for the first time, that GM-CSF is also a valid target for thetreatment of pain

SUMMARY OF THE INVENTION

The present invention, for the first time, demonstrates that GM-CSF is avalid target for the treatment of pain. This finding is new, and theprior art does not teach, suggest or provide any rational for such apoint of intervention in the treatment of pain. Accordingly, theinvention provides, e.g., a method for the treatment of pain in asubject, said method comprising the step of administering an effectiveamount of a GM-CSF antagonist to said subject.

In another aspect, the present invention contemplates a method for theprophylaxis of pain in a subject, said method comprising the step ofadministering an effective amount of GM-CSF antagonist to said subject.

In another aspect, the present invention is directed to a compositioncomprising a GM-CSF antagonist capable of antagonizing the ability ofGM-CSF from activating, proliferating, inducing growth and/or survivalof cells in a subject suffering from pain, or being suspected ofsuffering from pain, said composition further comprising one or morepharmaceutically acceptable carriers and/or diluents.

In another aspect, the present invention is directed to a compositioncomprising a GM-CSF antagonist useful in the treatment of pain, saidcomposition further comprising one or more pharmaceutically acceptablecarriers and/or diluents.

In particular aspects of the present invention, the GM-CSF antagonist isan antibody specific for GM-CSF.

In alternative aspects of the present invention, the GM-CSF antagonistis an antibody specific for the GM-CSF receptor.

In other aspects, the present invention is directed to the use of aGM-CSF antagonist in the preparation of a medicament in the treatment ofpain.

In other aspects, the present invention provides GM-CSF antagonists forthe treatment of pain.

In particular aspects of the present invention said pain ispost-surgical pain. In alternative aspects of the present invention saidpain is bone cancer pain. In yet alternative aspects of the presentinvention the GM-CSF antagonists have an analgesic effect.

In particular aspects of the present invention said pain is inflammatorypain.

In another aspect the present invention provides a geneticallyengineered mammal having a GM-CSF−/− genotype. In particular aspectssaid mammal is a mouse.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “have” and “include” and their respectivevariations such as “comprises”, “comprising”, “has”, “having”,“includes” and “including” will be understood to imply the inclusion ofa stated element or integer or group of elements or integers but not theexclusion of any other element or integer or group of elements orintegers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the knee joint histology scoring of the therapeutictreatment with a GM-CSF antibody in a mouse model of OA. Lat.=Lateral.Med.=Medial. Results are expressed as mean±SEM. For all areas, exceptthe Medial Femur, less disease was observed in mice treated withanti-GM-CSF antibody as compared to control mice.

FIG. 2 shows the result of an experiment assessing the hind limb weightdistribution in an incapacitance meter. Data are significant (unpairedt-test) from day 27 post OA induction onwards, as indicated in thegraph.

FIG. 3 shows the result of an experiment demonstrating the effectivenessof GM-CSF antagonists in the treatment of inflammatory disease(monoarticular arthritis induced by mBSA/IL1). Depicted are the clinicalscores of the knee joints at day 7 following treatment. Filled bars showthe results recorded for the C57BL/6 mice, open bars the results for theGM-CSF knock-out mice. Left: mice treated with indomethacin. Right: micewhich did not receive treatment with indomethacin.

FIG. 4 shows the result of an experiment demonstrating the effectivenessof GM-CSF antagonists in the treatment of inflammatory pain(monoarticular arthritis induced by mBSA/IL1). Shown is the weightdistribution measured in an incapacitance meter as a measure of pain inmice with mBSA/IL-1-induced monoarticular arthritis. Results areexpressed as injected limb/control limb*100.

FIG. 5 shows the result of an experiment demonstrating the effectivenessof GM-CSF antagonists in the treatment of inflammatory pain. Shown isthe level of inflammation as measured in the change in paw thicknessfollowing injection of CFA into the left (L) footpad. The right (R)footpad was normal. The “+ indomethacin” group was treated withindomethacin (1 mg/kg) i.p. 1 hr prior to each reading. N=12 mice/group.

FIG. 6 shows the result of an experiment demonstrating the effectivenessof GM-CSF antagonists in the treatment of inflammatory pain. Shown isthe weight distribution measured in an incapacitance meter as a measureof pain following injection of CFA into the left footpad. The rightfootpad was normal. The + indomethacin group was treated withindomethacin (1 mg/kg) i.p. 1 hr prior to each reading. Results areexpressed as injected limb/control limb×100. N=12 mice/group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention demonstrates that GM-CSF is a valid target for thetreatment of pain. In this respect, the invention provides, in oneaspect, methods of using a GM-CSF antagonist to bring about aprophylactic or therapeutic benefit in the field of pain.

The present invention provides therapeutic methods comprising theadministration of a therapeutically effective amount of a GM-CSFantagonist to a subject in need of such treatment. A “therapeuticallyeffective amount” or “effective amount”, as used herein, refers to theamount of a GM-CSF antagonist necessary to elicit the desired biologicalresponse. In accordance with the subject invention, the therapeuticeffective amount is the amount of a GM-CSF antagonist necessary to treatand/or prevent pain.

In certain aspects the present invention provides a method for thetreatment of post-surgical pain. In other aspects the present inventionprovides a method for the treatment of bone cancer pain. In yet otheraspects the present invention provides GM-CSF antagonists which have ananalgesic effect. In yet other aspects the present invention provides amethod for the treatment of rheumatoid arthritis pain. GM-CSFantagonists are capable of inhibiting or blocking the pain associatedwith rheumatoid arthritis. In other aspects the invention providesmethods for reducing incidence of rheumatoid arthritis pain,ameliorating rheumatoid arthritis pain, suppressing rheumatoid arthritispain, palliating rheumatoid arthritis pain, and/or delaying the onset,development, or progression of rheumatoid arthritis pain in a subject,said method comprising administering an effective amount of an GM-CSFantagonist to the subject. In another aspects the present inventionprovides a method for preventing or treating osteoarthritis pain in anindividual by administering an effective amount of an GM-CSF antagonistto the individual. In another aspect, the invention provides methods fortreating inflammatory cachexia (weight loss) associated with rheumatoidarthritis in an individual comprising administering an effective amountof an GM-CSF antagonist. In another aspect, the invention providesmethods for reducing incidence of osteoarthritis pain, amelioratingosteoarthritis pain, suppressing osteoarthritis pain, palliatingosteoarthritis pain, and/or delaying the onset, development, orprogression of osteoarthritis pain in an individual, said methodcomprising administering an effective amount of an GM-CSF antagonist tothe individual.

“Palliating” a pain or one or more symptoms of a pain (such asrheumatoid arthritis pain or osteoarthritis pain) means lessening theextent of one or more undesirable I clinical manifestations ofpost-surgical pain in an individual or population of individuals treatedwith an GM-CSF antagonist in accordance with the invention.

In certain aspects the pain is alleviated within about 24 hours afteradministering GM-CSF antagonist. In other aspects, the pain isalleviated within about 4 days after administering the GM-CSFantagonist.

“GM-CSF antagonists”, as used herein, includes GM-CSF antagonists in itsbroadest sense; any molecule which inhibits the activity or function ofGM-CSF, or which by any other way exerts a therapeutic effect on GM-CSFis included. The term GM-CSF antagonists includes, but is not limitedto, antibodies specifically binding to GM-CSF, inhibitory nucleic acidsspecific for GM-CSF or small organic molecules specific for GM-CSF. Alsowithin the meaning of the term GM-CSF antagonist are antibodiesspecifically binding to the GM-CSF receptor, inhibitory nucleic acidsspecific for the GM-CSF receptor or small organic molecules specific forthe GM-CSF receptor.

Inhibitory nucleic acids include, but are not limited to, antisense DNA,triplex-forming oligonucleotides, external guide sequences, siRNA andmicroRNA. Useful inhibitory nucleic acids include those that reduce theexpression of RNA encoding GM-CSF by at least 20, 30, 40, 50, 60, 70,80, 90 or 95 percent compared to controls. Inhibitory nucleic acids andmethods of producing them are well known in the art siRNA designsoftware is available.

Small organic molecules (SMOLs) specific for GM-CSF or the GM-CSFreceptor may be identified via natural product screening or screening ofchemical libraries. Typically the molecular weight of SMOLs is below 500Dalton, more typically from 160 to 480 Daltons. Other typical propertiesof SMOLs are one or more of the following:

-   -   The partition coefficient log P is in the range from −0.4 to        +5.6    -   The molar refractivity is from 40 to 130    -   The number of atoms is from 20 to 70

For reviews see Ghose et al, J Combin. Chem: 1:55-68, 1999 and Lipinskiet al, Adv Drug Del Rev: 23:3-25, 1997.

Preferably, a GM-CSF antagonist for use in the present invention is anantibody specific for GM-CSF or specific for the GM-CSF receptor. Suchan antibody may be of any type, such as a murine, a rat, a chimeric, ahumanized or a human antibody. A “human” antibody or functional humanantibody fragment is hereby defined as one that is not chimeric (e.g.,not “humanized”) and not from (either in whole or in part) a non-humanspecies. A human antibody or functional antibody fragment can be derivedfrom a human or can be a synthetic human antibody. A “synthetic humanantibody” is defined herein as an antibody having a sequence derived, inwhole or in part, in silico from synthetic sequences that are based onthe analysis of known human antibody sequences. In silico design of ahuman antibody sequence or fragment thereof can be achieved, forexample, by analyzing a database of human antibody or antibody fragmentsequences and devising a polypeptide sequence utilizing the dataobtained therefrom. Another example of a human antibody or functionalantibody fragment is one that is encoded by a nucleic acid isolated froma library of antibody sequences of human origin (i.e., such librarybeing based on antibodies taken from a human natural source).

A “humanized antibody” or functional humanized antibody fragment isdefined herein as one that is (i) derived from a non-human source (e.g.,a transgenic mouse which bears a heterologous immune system), whichantibody is based on a human germline sequence; or (ii) chimeric,wherein the variable domain is derived from a non-human origin and theconstant domain is derived from a human origin or (iii) CDR-grafted,wherein the CDRs of the variable domain are from a non-human origin,while one or more frameworks of the variable domain are of human originand the constant domain (if any) is of human origin.

The term “chimeric antibody” or functional chimeric antibody fragment isdefined herein as an antibody molecule which has constant antibodyregions derived from, or corresponding to, sequences found in onespecies and variable antibody regions derived from another species.Preferably, the constant antibody regions are derived from, orcorresponding to, sequences found in humans, e.g. in the human germ lineor somatic cells, and the variable antibody regions (e.g. VH, VL, CDR orFR regions) are derived from sequences found in a non-human animal, e.g.a mouse, rat, rabbit or hamster.

As used herein, an antibody “binds specifically to”, “specifically bindsto”, is “specific to/for” or “specifically recognizes” an antigen (here,GM-CSF or, alternatively, the GM-CSF receptor) if such antibody is ableto discriminate between such antigen and one or more referenceantigen(s), since binding specificity is not an absolute, but a relativeproperty. The reference antigen(s) may be one or more closely relatedantigen(s), which are used as reference points, e.g. IL3, IL5, IL-4,IL13 or M-CSF. In its most general form (and when no defined referenceis mentioned), “specific binding” is referring to the ability of theantibody to discriminate between the antigen of interest and anunrelated antigen, as determined, for example, in accordance with one ofthe following methods. Such methods comprise, but are not limited toWestern blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. Forexample, a standard ELISA assay can be carried out. The scoring may becarried out by standard color development (e.g. secondary antibody withhorseradish peroxide and tetramethyl benzidine with hydrogenperoxide).The reaction in certain wells is scored by the optical density, forexample, at 450 nm. Typical background (=negative reaction) may be 0.1OD; typical positive reaction may be 1 OD. This means the differencepositive/negative can be more than 10-fold. Typically, determination ofbinding specificity is performed by using not a single referenceantigen, but a set of about three to five unrelated antigens, such asmilk powder, BSA, transferrin or the like. Additionally, “specificbinding” may relate to the ability of an antibody to discriminatebetween different parts of its target antigen, e.g. different domains orregions of GM-CSF or the GM-CSF receptor, or between one or more keyamino acid residues or stretches of amino acid residues of GM-CSF or theGM-CSF receptor.

Also, as used herein, an “immunoglobulin” (Ig) hereby is defined as aprotein belonging to the class IgG, IgM, IgE, IgA, or IgD (or anysubclass thereof), and includes all conventionally known antibodies andfunctional fragments thereof. A “functional fragment” of anantibody/immunoglobulin hereby is defined as a fragment of anantibody/immunoglobulin (e.g., a variable region of an IgG) that retainsthe antigen-binding region. An “antigen-binding region” of an antibodytypically is found in one or more hypervariable region(s) of anantibody, i.e., the CDR-1, -2, and/or -3 regions; however, the variable“framework” regions can also play an important role in antigen binding,such as by providing a scaffold for the CDRs. Preferably, the“antigen-binding region” comprises at least amino acid residues 4 to 103of the variable light (VL) chain and 5 to 109 of the variable heavy (VH)chain, more preferably amino acid residues 3 to 107 of VL and 4 to 111of VH, and particularly preferred are the complete VL and VH chains(amino acid positions 1 to 109 of VL and 1 to 113 of VH; numberingaccording to WO 97/08320). A preferred class of immunoglobulins for usein the present invention is IgG. “Functional fragments” of the inventioninclude the domain of a F(ab′)₂ fragment, a Fab fragment, scFv orconstructs comprising single immunoglobulin variable domains or singledomain antibody polypeptides, e.g. single heavy chain variable domainsor single light chain variable domains. The F(ab′)₂ or Fab may beengineered to minimize or completely remove the intermoleculardisulphide interactions that occur between the C_(H1) and C_(L) domains.

An antibody of the invention may be derived from a recombinant antibodylibrary that is based on amino acid sequences that have been designed insilico and encoded by nucleic acids that are synthetically created. Insilico design of an antibody sequence is achieved; for example, byanalyzing a database of human sequences and devising a polypeptidesequence utilizing the data obtained therefrom. Methods for designingand obtaining in silico-created sequences are described, for example, inKnappik et a/, J. Mol. Biol. 296:57, 2000; Krebs et al, J. Immunol.Methods. 254:67, 2001, Rothe et al, J. Mol. Biol. 376:1182, 2008 andU.S. Pat. No. 6,300,064 issued to Knappik et al 2000 supra which herebyare incorporated by reference in their entirety.

Any antibody specific for GM-CSF may be used with the present invention.Exemplary antibodies are disclosed in U.S. Ser. No. 11/914,599, which isincorporated by reference in its entirety. Other exemplary antibodiesinclude antibodies comprising an amino acid sequence of a heavy chainvariable region as depicted in SEQ ID NO:1 or an amino acid sequence ofa light chain variable region as depicted in SEQ ID NO:2. Yet otherexemplary antibodies include antibodies which are derived fromantibodies comprising a heavy chain variable region as depicted in SEQID NO:1 or an amino acid sequence of a light chain variable region asdepicted in SEQ ID NO:2. Yet other exemplary antibodies includeantibodies which have the same specificity and/or bind to the sameepitope as antibodies comprising a heavy chain variable region asdepicted in SEQ ID NO:1 or an amino acid sequence of a light chainvariable region as depicted in SEQ ID NO:2. Yet other exemplaryantibodies include antibodies which comprise a heavy chain variableregion which is at least 70%, at least 80%, at least 90% or at least 95%homologous to the sequence depicted in SEQ ID NO:1. Yet other exemplaryantibodies include antibodies which comprise a light chain variableregion which is at least 70%, at least 80%, at least 90% or at least 95%homologous to the sequence depicted in SEQ ID NO.:2.

SEQ ID NO. 1: Met Glu Leu Ile Met Leu Phe Leu Leu Ser Gly ThrAla Gly Val His Ser Glu Val Gln Leu Gln Gln SerGly Pro Glu Leu Val Lys Pro Gly Ala Ser Val LysIle Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr AspTyr Asn Ile His Trp Val Lys Gln Ser His Gly LysSer Leu Asp Trp Ile Gly Tyr Ile Ala Pro Tyr SerGly Gly Thr Gly Tyr Asn Gln Glu Phe Lys Asn ArgAla Thr Leu Thr Val Asp Lys Ser Ser Ser Thr AlaTyr Met Glu Leu Arg Ser Leu Thr Ser Asp Asp SerAla Val Tyr Tyr Cys Ala Arg Arg Asp Arg Phe ProTyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr LeuArg Val Ser Ser Val Ser Gly Ser SEQ ID No. 2:Met Gly Phe Lys Met Glu Ser Gln Ile Gln Val PheVal Tyr Met Leu Leu Trp Leu Ser Gly Val Asp GlyAsp Ile Val Met Ile Gln Ser Gln Lys Phe Val SerThr Ser Val Gly Asp Arg Val Asn Ile Thr Cys LysAla Ser Gln Asn Val Gly Ser Asn Val Ala Trp LeuGln Gln Lys Pro Gly Gln Ser Pro Lys Thr Leu IleTyr Ser Ala Ser Tyr Arg Ser Gly Arg Val Pro AspArg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe IleLeu Thr Ile Thr Thr Val Gln Ser Glu Asp Leu AlaGlu Tyr Phe Cys Gln Gln Phe Asn Arg Ser Pro LeuThr Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys ArgAla Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Lys Gly Glu Phe

Alternative exemplary antibodies that can be used in the presentinvention are antibodies comprising an amino acid sequence of a heavychain variable region as depicted in SEQ ID NO:3 or an amino acidsequence of a light chain variable region as depicted in SEQ ID NO:4.Other exemplary antibodies include antibodies which are derived fromantibodies comprising a heavy chain variable region as depicted in SEQID NO:3 or an amino acid sequence of a light chain variable region asdepicted in SEQ ID NO:4. Yet other exemplary antibodies includeantibodies which have the same specificity and/or bind to the sameepitope as antibodies comprising a heavy chain variable region asdepicted in SEQ ID NO:3 or an amino acid sequence of a light chainvariable region as depicted in SEQ ID NO:4. Yet other exemplaryantibodies include antibodies which comprise a heavy chain variableregion which is at least 70%, at least 80%, at least 90% or at least 95%homologous to the sequence depicted in SEQ ID NO:3. Yet other exemplaryantibodies include antibodies which comprise a light chain variableregion which is at least 70%, at least 80%, at least 90% or at least 95%homologous to the sequence depicted in SEQ ID NO:4.

SEQ ID NO. 3: heavy MORQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMNWVRQAPGKGLEWVSGIENKYAGGATYYAASVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARGFGTDFWGQGTLVTVSSSEQ ID NO. 4: light MORDIELTQPPSVSVAPGQTARISCSGDSIGKKYAYWYQQKPGQAPVLVIYKKKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCSAWGDKGMVFGG GTKLTVLGQ

Alternative exemplary antibodies that can be used in the presentinvention are antibodies comprising a H-CDR3 sequence selected from:

(SEQ ID NO. 5) Ser Gly Leu Ile Phe Asp Tyr Trp Leu Asp,1               5                   10 (SEQ ID NO. 6)Ser Gly Leu Ile Ile Asp Ala Leu Ser Pro,1               5                   10 (SEQ ID NO. 7)Thr Ser Leu Met Ser Ile Tyr Phe Asp Tyr,1               5                   10 (SEQ ID NO. 8)Ser Gly Leu Leu Phe Leu Tyr Phe Asp Tyr,1               5                   10 (SEQ ID NO. 9)Ser Gly Leu Ile Asn Leu Gly Met His Pro,1               5                   10 (SEQ ID NO. 10)Ser Gly Leu Ile Phe Asp Ala Leu Arg Asp,1               5                   10 (SEQ ID NO. 11)Ser Gly Leu Ile Phe Asp Lys Leu Thr Ser,1               5                   10 (SEQ ID NO. 12)Ser Gly Leu Ile Asn Leu His Phe Asp Thr,1               5                   10 (SEQ ID NO. 13)Ser Thr His Phe Ser Ala Tyr Phe Asp Tyr,1               5                   10 (SEQ ID NO. 14)Ser Gly Leu Ile Met Asp Lys Leu Asp Asn,1               5                   10 (SEQ ID NO. 15)Ser Gly Leu Ile Ile Asp Asn Leu Asn Pro,1               5                   10 and (SEQ ID NO. 16)Ser Gly Leu Ile Ala Val Tyr Phe Asp Tyr.1               5                   10

Preferably, the antibodies comprising a H-CDR3 sequence selected fromany one of SEQ ID NOs. 5-16, additionally comprise the following H-CDR1sequence:

Asp Tyr Leu Leu His, (SEQ ID NO. 17)  1               5and/or the following H-CDR2 sequence:

(SEQ ID NO. 18) Trp Leu Asn Pro Tyr Ser Gly Asp Thr Asn1               5                   10  Tyr Ala Gln Lys Phe Gln Gly,                15and/or the following L-CDR1 sequence:

(SEQ ID NO. 19) Arg Ala Ser Gln Asn Ile Arg Asn Ile Leu Asn,1               5                   10and/or the following L-CDR2 sequence:

(SEQ ID NO. 20) Ala Ala Ser Asn Leu Gln Ser, 1               5and/or the following L-CDR3 sequence:

(SEQ ID NO. 21) Gln Gln Ser Tyr Ser Met Pro Arg Thr. 1               5

Alternative exemplary antibodies that can be used in the presentinvention are antibodies comprising the following L-CDR1 sequence:

(SEQ ID NO. 22) Arg Ala Ser His Arg Val Ser Ser Asn Tyr Leu Ala,1                5                   10and/or the following L-CDR2 sequence:

(SEQ ID NO. 23) Gly Ala Ser Asn Arg Ala Thr, 1                5and/or the following L-CDR3 sequence:

(SEQ ID NO. 24) Gln Gln Tyr Ala Ser Ser Pro Val Thr, 1                5and/or the following H-CDR1 sequence:

(SEQ ID NO. 25) Gly Tyr Ile Phe Pro Thr Phe Ala Leu His,1                5                   10and/or the following H-CDR2 sequence:

(SEQ ID NO. 26) Ser Ile Asn Thr Ala Ser Gly Lys Thr Lys1                5                   10  Phe Ser Thr Lys Phe Gln,                15and/or the following H-CDR3 sequence:

(SEQ ID NO. 27) Asp Arg Phe Gln Asn Ile Met Ala Thr Ile1               5                   10 Leu Asp Val.

Preferably said antibody comprise all the CRDs of SEQ ID NOs. 22-27.

The GM-CSF receptor is a member of the haematopoietin receptorsuperfamily. It is heterodimeric, consisting of an alpha and a betasubunit. The alpha subunit is highly specific for GM-CSF whereas thebeta subunit is shared with other cytokine receptors, including IL3 andIL5. This is reflected in a broader tissue distribution of the betareceptor subunit. The alpha subunit, GM-CSFR α, is primarily expressedon myeloid cells and non-haematopoetic cells, such as neutrophils,macrophages, eosinophils, dendritic cells, endothelial cells andrespiratory epithelial cells. Full length GM-CSFR α is a 400 amino acidtype I membrane glycoprotein that belongs to the type I cytokinereceptor family, and consists of a 22 amino acid signal peptide(positions 1-22), a 298 amino acid extracellular domain (positions23-320), a transmembrane domain from positions 321-345 and a short 55amino acid intra-cellular domain. The signal peptide is cleaved toprovide the mature form of GM-CSFR α as a 378 amino acid protein. cDNAclones of the human and murine GM-CSFR α are available and, at theprotein level, the receptor subunits have 36% identity. GM-CSF is ableto bind with relatively low affinity to the α subunit alone (Kd 1-5 nM)but not at all to the β subunit alone. However, the presence of both αand β subunits results in a high affinity ligand-receptor complex(Kd>>100 pM). GM-CSF signalling occurs through its initial binding tothe GM-CSFR α chain and then cross-linking with a larger subunit thecommon β chain to generate the high affinity interaction, whichphosphorylates the JAK-STAT pathway.

Any antibody specific for GM-CSF receptor may be used with the presentinvention. Exemplary antibodies include antibodies comprising an aminoacid sequence of a H-CDR3 sequence depicted in any one of SEQ IDNo's.:28-46. Other exemplary antibodies include antibodies which arederived from antibodies comprising an amino acid sequence of a H-CDR3sequence depicted in any one of SEQ ID No's.:28-46. Yet other exemplaryantibodies include antibodies which have the same specificity and/orbind to the same epitope as antibodies comprising an amino acid sequenceof a H-CDR3 sequence depicted in any one of SEQ ID NOs.:28-46. Yet otherexemplary antibodies include antibodies which comprise a H-CDR3 sequencewhich is at least 70%, at least 80%, at least 90% or at least 95%homologous to the H-CDR3 sequence depicted in any one of SEQ IDNOs.:28-46.

SEQ ID NO: 28: Val Gly Ser Phe Ser Gly Ile Ala Tyr Arg Pro                5                   10 SEQ ID NO: 29:Val Gly Ser Phe Ser Gly Pro Ala Leu Arg Pro                5                   10 SEQ ID NO: 30:Val Gly Ser Phe Ser Pro Pro Thr Tyr Gly Tyr                5                   10 SEQ ID NO: 31:Val Gly Ser Phe Ser Gly Tyr Pro Tyr Arg Pro                5                   10 SEQ ID NO: 32:Val Gly Ser Phe Ser Pro Leu Thr Leu Gly Leu                5                   10 SEQ ID NO: 33:Val Gly Ser Phe Ser Gly Pro Val Tyr Gly Leu                5                   10 SEQ ID NO: 34:Val Gly Ser Phe Ser Pro Pro Ala Tyr Arg Pro                5                   10 SEQ ID NO: 35:Val Gly Ser Phe Ser Pro Val Thr Tyr Gly Leu                5                   10 SEQ ID NO: 36:Val Gly Ser Phe Ser Gly Leu Ala Tyr Arg Pro                5                   10 SEQ ID NO: 37:Val Gly Ser Phe Ser Pro Ile Thr Tyr Gly Leu                5                   10 SEQ ID NO: 38:Val Gly Ser Phe Ser Gly Trp Ala Phe Asp Tyr                5                   10 SEQ ID NO: 39;Val Gly Ser Phe Ser Gly Trp Ala Phe Asp Tyr                5                   10 SEQ ID NO: 40:Leu Gly Ser Val Thr Ala Trp Ala Phe Asp Tyr                5                   10 SEQ ID NO: 41:Ala Gly Ser Ile Pro Gly Trp Ala Phe Asp Tyr                5                   10 SEQ ID NO: 42:Val Gly Ser Phe Ser Pro Leu Thr Met Gly Leu                5                   10 SEQ ID NO: 43:Val Gly Ser Phe Ser Pro Leu Thr Met Gly Leu                5                   10 SEQ ID NO: 44:Val Gly Ser Phe Ser Gly Pro Ala Leu His Leu                5                   10 SEQ ID NO: 45:Val Gly Ser Val Ser Arg Ile Thr Tyr Gly Phe                5                   10 SEQ ID NO: 46:Val Gly Ser Phe Ser Pro Leu Thr Leu Gly Leu                5                   10

In certain aspects, the present invention provides methods for thetreatment of pain in a subject, said method comprising the step ofadministering a GM-CSF antagonist to said subject. “Subject”, as used inthis context refers to any mammal, including rodents, such as mouse orrat, and primates, such as cynomolgus monkey (Macaca fascicularis),rhesus monkey (Macaca mulatta) or humans (Homo sapiens). Preferably thesubject is a primate, most preferably a human.

In certain aspect the present invention provides a compositioncomprising a GM-CSF antagonist capable of antagonizing the ability ofGM-CSF from activating, proliferating, inducing growth and/or survivalof cells in a subject suffering from pain, or being suspected ofsuffering from pain, said composition further comprising one or morepharmaceutically acceptable carriers and/or diluents. Anti-GM-CSFantibodies of the present invention may antagonize any of the roles ofGM-CSF in pain.

In another aspect the present invention provides a method for theprophylaxis of pain in a subject, said method comprising administering aGM-CSF antagonist to said subject. “Prophylaxis” as used in this contextrefers to methods which aim to prevent the onset of a disease or whichdelay the onset of a disease.

In certain aspect the present invention provides a compositioncomprising a GM-CSF antagonist useful in the treatment of pain, saidcomposition further comprising one or more pharmaceutically acceptablecarriers and/or diluents.

In other aspects the present invention provides the use of a GM-CSFantagonist in the preparation of a medicament in the treatment of pain.

In other aspects the present invention provides GM-CSF antagonists forthe treatment of pain.

The compositions of the present invention are preferably pharmaceuticalcompositions comprising a GM-CSF antagonist and a pharmaceuticallyacceptable carrier, diluent or excipient, for the treatment of pain.Such carriers, diluents and excipients are well known in the art, andthe skilled artisan will find a formulation and a route ofadministration best suited to treat a subject with the GM-CSFantagonists of the present invention.

In another aspect the present invention provides a geneticallyengineered mammal having a GM-CSF−/− genotype. In particular aspectssaid mammal is a mouse. The terms “knock-out” mouse (or mammal), a mouse(or mammal) “disrupted in” a certain gene, and a mouse (or mammal) witha “−/− genotype” are used interchangeably in the present invention andare art recognized. Respective animals are deficient in a respectivegene, here GM-CSF, on both alleles of the chromosome.

Example 1 Generation of a GM-CSF−/− Mouse

The generation of GM-CSF−/− mice is described in Stanley et al (1994).Proc. Natl. Acad. Sci. USA 91:5592. Briefly, chimeric mice weregenerated by microinjection of 129/OLA-derived ES cells (H-2b) with adisrupted GM-CSF gene into C57BL/6 (H-2b) host blastocysts. Germlinetransmitters of the mutated GM-CSF allele were crossed with C57BL/6 micefor 11 generations, giving GM-CSF+/− mice that were interbred to yieldthe GM-CSF−/−, GM-CSF+/−, and GM-CSF+/+ mice used for the experiments.GM-CSF genotype status was determined by PCR analysis of tail DNA.Animals were fed standard rodent chow and water ad libitum and werehoused with same sex littermates in sawdust-lined cages. Mice of bothsexes were consigned to experiments at 8 to 15 wk of age

Example 2 GM-CSF Antagonists are Effective in Treating Post-SurgicalPain

A pain model is used that mimics post surgical pain to assess theefficacy of treatment with GM-CSF antagonists.

Animals:

Male Sprague Dawley rats weighting between 220-240 grams are acclimatedto the animal facility for one week prior to surgery.

Surgery:

The surgery is based on the procedure described in Brennan et al, Pain64:493-501, 1996. Animals are anesthetized with a 2% isoflurane in airmixture that is maintained during surgery via a nose cone. The plantersurface of the right hind paw is prepared with a povidone-iodine pad,and a 1-cm central longitudinal incision is made through skin andfascia, starting 0.5 cm from the edge of the heel and extending towardthe toes. Measurements are made with a ruler with the foot held in aflexed position. The plantaris muscle is elevated using curved forcepsand incised longitudinally. The muscle is incised through its fulldepth, between the origin and insertion. Bleeding is controlledthroughout surgery by pressure applied through a gauze pad. The wound isclosed with two mattress sutures (5-0 ethilon black monofilament). Thesesutures are knotted 5-6 times, with the first knot loosely tied. Thewound site is swabbed with bacitracin solution. Animals are allowed torecover and rest in clean cages for two hours or more before behavioraltesting began.

Evaluation of Resting Pain:

A cumulative pain score is used to assess pain related to weightbearing. Animals are placed on a plastic mesh (grid: 8 mm2) in clearplastic cages that are elevated on a platform (h: 18″) allowinginspection of the underside of their paws. After a 20 minute acclimationperiod, weight bearing is assessed on a scale of 0 to 2. A score of 0 isgiven if the paw is blanched or pressed against the mesh, indicatingfull weight bearing. A score of 1 is given if the paw is favored withthe skin just touching the mesh, with no blanching or indentation of theskin. A score of 2 is given if the paw is held completely off the mesh.Flinching the paw is considered a 2 if the rat is still at rest. Eachanimal is observed for 1 minute every 5 minutes for minutes. The sum of6 scores (0-12) obtained during ½-hour is used to assess pain in theincised foot. Frequency of scores of 2 is also calculated and used toassess the incidence of severe pain or total guarding of the paw by theanimal. Each animal is tested 24 hours before surgery (baseline), and 2h, 24 h, 48 h, and 72 h postoperatively. The results of this experimentshow that the cumulative resting pain score observed in animals treatedwith GM-CSF antagonists is significantly reduced compared to controlanimals. Weight bearing is a good correlate of how willing the animal isto use the limb, and therefore is an effective measure of pain relief.Preferably, the GM-CSF antagonist is an antibody specific for GM-CSF ofspecific for the GM-CSF receptor. Such antibodies are injected intraperitoneal (i.p.) at various concentrations of the antibody (e.g. 0.004,0.01, 0.02, 0.1, 0.6, and 1 mg per kilogram of animal weight) at 15hours pre-incision. The negative control group receives no antibody butis injected i.p. with a saline solution. Fentanyl at 0.01 mg/kg isinjected i.p. as a positive control 30 minutes before testing at 24hours post-surgery. Each experiment involves 8 animals (n=8 per group)for each condition, and the control group has 56 animals. Surgery isperformed and a cumulative pain score is measured as described above.Resting pain is evaluated twenty-four hours after the surgery.

GM-CSF antagonists significantly reduce resting pain after surgery whenadministered at 0.02 mg/kg to 1 mg/kg dosage.

In another experiment, the efficacy of GM-CSF antagonists in reducingpost-surgical pain when administered post-surgically is tested. GM-CSFspecific or GM-CSF receptor specific antibodies are injectedintravenously (i.v.) two hours after surgery. The control group receivesno antibody but was injected i.v. with a saline solution. Surgery isperformed and resting pain expressed as a cumulative pain score isassessed 24 hours after surgery. Treatment with GM-CSF antagonistsignificantly reduces resting pain at twenty-four hours after incisionwhen the antibody is administered 2 hours post-incision. These resultsdemonstrated that GM-CSF antagonist effectively alleviated post-surgicalpain when administered after surgery.

Evaluation of Thermal Hyperalgesia:

Thermal hyperalgesia is assessed by the rat planter test (Ugo Basile,Italy) following a modified method of Hargreaves et al. (1988). Rats arehabituated to the apparatus that consisted of four individual plexiglassboxes on an elevated glass table. A mobile radiant heat source islocated under the table and focused onto the hind paw. While the animalis still, but not sleeping, the button on the control box is depressed,the radiant heat source comes on and the time taken for the animal towithdraw from the heat source is automatically recorded. This pawwithdrawal latency (POOL) is detected by a light detector embedded in,the radiant heat source that senses the movement of the rat paw by achange in reflectance of the radiant source. Paw Withdrawal Latencies(PWL), in seconds, were recorded: There is an automatic cut-off point of22.5 s to prevent tissue damage. PWL are taken three to four times forboth hind paws of each animal, the mean of which represent base linesfor right and left hind paws. The results are presented as the ratio ofscore measured in the right paw (site of surgery) and the left paw. Theapparatus is calibrated once (at the beginning of the study) and set tointensity of 40 to give a normal PWL of approximately 6 seconds. Eachanimal is tested 24 hours before surgery (baseline), and 3 h, 24 h, 48h, and 72 h postoperatively. Thermal hyperalgesia measurements are takenafter tactile allodynia measurements. The results demonstrated thattreatment with GM-CSF antagonists significantly reduced post-surgicalthermal hyperalgesia.

Example 3 GM-CSF Antagonists are Effective in Treating Bone Cancer Pain

GM-CSF antagonists, such as GM-CSF specific antibodies or GM-CSFreceptor specific antibodies are effective in treating cancer painassociated with bone metastasis.

We use a murine bone cancer pain model to assess the efficacy oftreatment with GM-CSF antagonists. This murine model of bone cancer painis developed by intramedullary injection of osteolytic sarcoma cellsinto the mouse femur and the needle hole is then filled with dentalamalgam to confine the tumor to bone (see Schwei et al, J: Neuroscience19:10886-10897, 1999 and Luger et al, Pain 99:397-406, 2002).Experiments are performed on adult male C3H/HeJ mice. On day 0, anarthrotomy is performed following induction of general anesthesia withsodium pentobarbital (50 mg/kg, intraperitoneal (i.p.)). A needle isinserted into the medullary canal to create a pathway for the sarcomacells. A depression is then made using a pneumatic dental high speedhandpiece. In addition to naive animals (n=5), sham animals (n=5) aregenerated with an injection of a minimum essential, media (20 μl, Sigma,St. Louis, Mo.) into the intramedullary space of the femur (designatedsham) whereas sarcoma animals (n=5 for each condition tested) areinjected with media containing 105 2472 osteolytic sarcoma cells(designated sarcoma or sarc) (20 μl, ATCC, Rockville, Md.). For allanimals, the injection site is sealed with a dental amalgam plug toconfine the cells or injected media within the intramedullary canal andfollowed by irrigation with sterile water (hypotonic solution). Finally,incision closure is achieved with wound clips. Clips are removed at day5 so as not to interfere with behavioral testing. A second group ofsarcoma-injected animals is treated with GM-CSF specific or GM-CSFreceptor specific antibodies (e.g. 10 mg/kg, i.p.) on days 6 and 13.

Behavioral Analysis:

Animals are tested for pain-related behaviors on day 10 and day 14post-tumor implantation. Animals are behaviorally tested using thefollowing tests: ongoing pain (spontaneous guarding and flinching),ambulatory pain (limb use and rotarod), and movement-evoked pain(palpation-evoked guarding and palpation-evoked flinching). Animals areplaced in a clear plastic observation box with a wire mesh floor and areallowed to habituate for a period of 30 min. After acclimation,spontaneous guarding, spontaneous flinching, limb use during normalambulation in an open field, and guarding during forced ambulation isassessed. Palpation-induced guarding and flinching are measured afterthe 2 min period of normally non-noxious palpation of the distal femurin sarcoma- and sham-injected animals.

The number of spontaneous flinches and time to spent guarding,representative of nociceptive behavior, are recorded simultaneouslyduring a 2-min observation period. Guarding is defined as the time thehindpaw is held aloft while ambulatory and flinches are the number oftimes the animal held the limb aloft. Normal limb use during spontaneousambulation is scored on a scale of 5 to 0: (5) normal use, and (0)complete lack of limb use.

Forced ambulatory guarding is determined using a rotarod (ColumbusInstruments, Columbus, Ohio). The rotated machine has a revolving rodand is equipped with speed, acceleration, and sensitivity controls. Theanimals are placed on the rod with ×4 speed, 8.0 acceleration, and 2.5sensitivity. Forced ambulatory guarding is rated on a scale of 5-0: (5)normal use, and (0) complete lack of use. After a normally non-noxiouspalpation of the distal femur in animals every second for 2 min, theanimals are placed in the observation box and their palpation-inducedguarding and palpation-induced flinching is measured for an additional 2min.

Treatment with GM-CSF Antagonists:

On day 6 and day 13, sarcoma-injected animals are intraperitoneally(i.p.) injected with GM-CSF antagonists, such as an anti-GM-CSF or ananti-GM-CSF receptor antibody (n=5), or sarcoma- and sham-injectedanimals were injected (i.p.) with saline (n=5 for each condition). Allanimals are behaviorally analyzed on days 10 and 14.

Evaluation of Ongoing Pain Behaviors:

Sarcoma-injected animals (administered with saline) developstatistically significant ongoing pain behaviors, as assessed byspontaneous guarding and spontaneous, as compared to sham injectedanimals (administered with saline).

Administration of GM-CSF antagonists significantly reduce spontaneousguarding and spontaneous flinching in sarcoma-injected mice on day 10and day 14 post-sarcoma implantation as compared to administration ofsaline to sarcoma-injected mice. These results indicate that GM-CSFantagonists reduce ongoing pain in sarcoma-injected mice.

Evaluation of Ambulator Pain Behaviors:

Sarcoma-injected animals (administered with saline) develop ambulatorypain behaviors as assessed by limb use and forced ambulation guarding(rotarod), as compared to sham-injected animals (administered withsaline). Administration of GM-CSF antagonists significantly increaseslimb use score and forced ambulatory guarding score in sarcoma-injectedmice on day 10- and day 14 post-sarcoma implantation, as compared toadministration of saline to sarcoma-injected mice. These resultsindicate that GM-CSF antagonists reduce ambulatory pain insarcoma-injected mice.

Evaluation of Touch-Evoked Pain Behaviors:

Sarcoma injected animals (administered with saline) develop touch-evokedpain behaviors as assessed by palpation-induced guarding andpalpation-induced flinching, as compared to sham-injected animals(administered with saline). Administration of GM-CSF antagonistssignificantly reduces palpation-induced guarding and palpation-inducedflinching in sarcoma-injected mice on day 10 and day 14 post-sarcomaimplantation as compared to administration of saline to sarcoma-injectedmice. These results indicate that GM-CSF antagonists reduce touch-evokedpain in sarcoma-injected mice.

Example 4 Analgesic Effects of GM-CSF Antagonists

The analgesic effects of GM-CSF antagonists in complete Freund'sadjuvant (CFA)-induced chronic arthritis in rats is investigated usingthe vocalization test, in comparison with indomethacine used asreference substance.

Fifty (50) male Lewis rats (LEWIS LEW/Crl Ico) weighing 150 g to 220 gat the beginning of the experimental phase are included in this study.All animals are kept for at least 5 days before the experiment, and arehoused in a temperature (19.5-24.5° C.), relative humidity (45-65%) and12-h light/dark cycle controlled room with ad libitum access to filteredtap-water and standard pelleted laboratory chow throughout the study.Animals are individually identified on the tail.

On day 0 (D0), arthritis is induced in rats by intradermal injectioninto the tail of 0.05 ml of a Mycobacterium butyricum suspension Inmineral oil (10 mg/ml). On day 14 (D14), arthritic rats are included inthe study according to their ability to vocalize upon gentle flexion ofthe hindpaw and by their arthritis index, evaluated using aninflammation score for each hind and forepaw (see Kuzuna et al, Chem.Pharm. Bull. (Tokyo) 23:1184-1191, 1975 and Pearson et al, ArthritisRheum. 2:440-459, 1959).

Animals are scored based on the following criteria: Score 0: normalaspect; Score 1: erythema; Score 2: erythema with slight edema; Score 3:strong inflammation without ankylosis; Score 4: ankylosis. Only animalsable to vocalize upon gentle flexion and presenting a score of 2 or 3are included in the study.

Four groups of 10 rats each are included in the study. For group 1(vehicle), on day 14 (D14), after selection, rats are intravenouslyadministered by vehicle (saline). On day 18 (D18), the nociceptiveintensity is evaluated by gentle flexion of the hindpaw and theintensity of the level of vocalization is recorded for each animal. Forgroup 2 (4 days), on D 14, after selection, rats are intravenouslyadministered GM-CSF-specific antibody. On day 18 (D18), the nociceptiveintensity is evaluated by gentle flexion of the hindpaw and theintensity of the level of vocalization is recorded for each animal. Forgroup 3 (24 hours), on day 17 after injection of CFA, rats areintravenously administered GM-CSF-specific antibody or GM-CSFreceptor-specific antibody. The nociceptive intensity is evaluated bygentle flexion of the hindpaw 24 hours later, and the intensity of thelevel of vocalization is recorded for each animal. For group 4(indomethacin), on day 18 (D18), the nociceptive intensity is evaluatedby gentle flexion of the hindpaw one hour after oral administration ofindomethacin (10 mg/kg). The intensity of the level of vocalization isalso recorded for each animal. The test substances are administered in ablind and random manner by intravenous route under a volume of 5 ml/kg,whereas indomethacin was administered by oral route under a volume of 10ml/kg.

GM-CSF antagonists show an significant analgesic effects. Statisticalsignificance between the treated groups and the vehicle group aredetermined with a Dunnett's test using the residual variance after aone-way analysis of variance. GM-CSF-specific antibody and GM-CSFreceptor-specific antibody significantly reduces pain in a rat model ofrheumatoid arthritis 24 hours or 4 days after a single administration ofthe antibody.

Example 5 GM-CSF Antagonists are Effective in Treating OsteoarthriticPain

In this experiment we used a monoclonal antibody specific for GM-CSF todemonstrate that a GM-CSF antagonist can be effective to treatosteoarthritic pain.

Collagen-Induced OA Mouse Model:

C57BL/6 mice were given 1 unit of collagenase type VII intra-articularlyinto the right knee on days 0 and 2 to induce joint instability (seeBlom et al. (2004) Osteoarthritis Cartilage. 12; 627-35).

Anti-GM-CSF Antibody Treatment:

20 mice were randomly divided into 2 groups (10 mice/group).

Group 1 (n=10): anti-GM-CSF antibody (22E9)

Group 2 (n=10): IgG2a isotype control antibody.

Mice were treated intraperitoneally, three times per week for 6 weekswith 250 μg/mouse/treatment anti-GM-CSF antibody (22E9) or IgG2aisoptype control antibody. Treatment started 4 days before the inductionof OA (prophylactic), i.e. mice were treated on day −4, day −2, day 0(the day of the first collagenase injection), then 3 times per weekuntil the end of the experiment at 6 weeks). At weeks 2, 4 and 6, micewere bled. Both, the control antibody and the anti-GM-CSF antibody werepurified to contain less than 10 Endotoxin Units/ml.

The antibody 22E9 was used as an exemplary anti-GM-CSF antibody. 22E9,which is of IgG2a isotype, is a rat anti-mouse GM-CSF-specific antibody.22E9 was purchased from AbD Serotec (Martinsried, Germany; Cat. No.1023501). Alternative suppliers exist, e.g. eBioscience (SanDiego,Calif., USA, Cat. No. 14-7331).

Histology:

6-weeks post final injections, histology was performed on the mice kneejoints. The knee joints were collected, fixed, de-calcified, embedded inparaffin and cut at 7 μm with a microtome. Slides were stained withSafranin-O/Fast Green and Haematoxylin and Eosin to demonstrate jointpathology. Pathology investigated included: cartilage damage, synovitis,osteophyte formation and joint deformation.

The scoring system used for cartilage pathology was as follows:

Grade

0 Normal

1 Irregular but intact

1.5 Irregular with rough surface

2 Superficial fibrillation

2.5 Superficial fibrillation with reduced cells in cartilage layer

3 Vertical fissures

3.5 Branching and/or horizontal assures, tidemark ruptures

4 Cartilage loss not extending to the tide mark

4.5 Cartilage loss extending to the tide mark

5 Cartilage loss beyond the tide mark but not extending to the bone

5.5 Cartilage loss extending to the bone

6 Bone loss/remodeling/deformation

Stage

1 <10% area damaged

2 10-25% area damaged

3 25-50% area damaged

4 50-75% area damaged

The grade was multiplied by the stage to give the score.

This scoring system is based on a recognized method to assess OAhistopathology in clinical and experimental OA. See Pritzker et al.(2006) Osteoarthritis Cartilage; 14; 13-29. Grade is defined as OA depthprogression into cartilage. Stage is defined as the horizontal extent ofcartilage involvement, i.e. how much of the cartilage is affected. Gradeis multiplied by the stage to give the score to give an overall score,so as to represent a combined assessment of OA severity and extent. Upto six sections are scored per mouse.

Grade was multiplied by the stage to give the score.

The following scoring system was used for synovitis (Synovial layerscoring system):

-   -   0 No changes compared to normal joints    -   1 Thickening of the synovial lining and some influx of        inflammatory cells    -   2 Thickening of the synovial lining and intermediate influx of        inflammatory cells    -   3 Profound thickening of the synovial lining and maximal        observed influx of inflammatory cells        Pain Measurements:

An indicator of pain used for OA models is differential distribution ofweight measured using an Incapacitance Meter. This instrument measureschanges in weight distribution between the operated and contralateral,unoperated hind limb. Mice were allowed to acclimatize to the equipmenton three occasions prior to the experiment. Weight placed on each hindlimb was measured over a 5 second period. Three separate measurementstaken per mouse for each time point then averaged. Measurements wereperformed 2 times per week throughout the experiment. Results areexpressed as collagenase injected limb/control limb×100.

Results:

For all areas analyzed in histology (except the Medial Femur), i.e. theLateral Femur, the Lateral Tibia, and the Medial Tibia, there was aclear trend towards less disease in mice treated with anti-GM-CSFantibody. Results are depicted in FIG. 1.

Assessment of the weight distribution, as a measure of pain associatedwith the arthritis, showed a significant shift in weight away from thearthritic knee from day 27 onwards in the anti-GM-CSF mAb-treated groupcompared to the control mAb-treated group. Results are depicted in FIG.2.

Mice treated with a GM-CSF antagonist showed less disease as compared tomice treated with the control antibody. Mice treated with the GM-CSFantagonist also showed significantly less pain in the latter stages ofdisease compared to mice treated, with the control antibody. Micetreated with the isotype control antibody showed significant increasedsigns of osteoarthritis as compared to the mice which received theGM-CSF-specific antibody. This demonstrates that GM-CSF antagonists areeffective in the treatment of osteoarthritic pain.

Example 6 GM-CSF Antagonists are Effective in Treating InflammatoryPain/mBSA Model

The following experiment demonstrates that GM-CSF antagonists are alsoeffective in the treatment of inflammatory pain. To do so, mBSA/IL-1monoarticular arthritis was induced in GM-CSF knock-out mice and incontrol mice. Pain was assessed with or without administration ofindomethacin, a pain relieving substance, at various time points usingan incapacitance tester.

Mice

24 male C57BL/6 mice and 24 male GM-CSF−/− mice (see Example 1) wereused in four treatment groups:

Group 1: GM-CSF KO (n=12): methylated BSA/IL-1

Group 2: GM-CSF KO (n=12): methylated mBSA/IL-1+ indomethacin

Group 3: C57BL/6 wildtype (n=12): methylated BSA/IL-1

Group 4: C57BL/6 wildtype (n=12): methylated BSA/IL-1+ indomethacin

Induction of Monoarticular Arthritis

Monoarticular arthritis was induced by intraarticular injection of 10 μlof mBSA (20 mg/ml) in saline into the knee joint and 10 μl of salineinto the contralateral knee joint. 20 μl of IL-1β (250 ng) wassubcutaneously administered daily for 3 days. A response typicallydevelops between days 4 and 7 after injection of mBSA and resolves byday 28. Incapacitance was tested on days 2, 3, 4, 5 and 7.

Indomethacin (Sigma) is a non-steroidal anti-inflammatory drug commonlyused to reduce fever, pain, stiffness, and swelling. It works byinhibiting the production of prostaglandins. 1 mg/kg i.p. indomethacinwas administered to groups 2 and 4 one hour before pain was assessedusing a capacitance meter.

Read Out for Pain

An Incapacitance Tester (Dual Weight Averager) was used to automaticallyand reproducibly assess the analgesic potency by measuring the weightdistribution on the two hind paws. The force exerted by each limb(measured in grams) is averaged over a user selectable period thusindicating any tendency for the animal to shift its weight from one sideto another, hence providing a quantitative measurement of incapacitance.

Weight placed on each hind limb was measured over a 5 second period. 3separate measurements taken per mouse for each time point then averaged.Results are expressed as injected limb/control limb×100. Thus a value of100 means that equal weight is being placed on the right and the leftlimb. A value below 100 means less weight is being placed on theinjected limb (left) compared with the control limb (right).

Results

This model induces synovitis in the knee joint via the injection ofmBSA. At day 7, the knee joints were examined visually and given a scorefrom 0 (normal) to 3 (severely inflamed) (FIG. 3). The left knee, whichwas injected with mBSA, was significantly more inflamed compared withthe right knee (injected with saline) (p>0.001 for C57 mice and p=0.02for GM-CSF−/− mice) (FIG. 3). In fact, all right knees, which wereinjected with saline, received a score of 0. There was no significantdifferences between mice treated with indomethacin and those not foreither strain.

C57BL/6 mice showed significantly more pain (as measured by a shift inweight away from the mBSA-injected knee) compared to GM-CSF−/− mice whenmBSA/IL-1 monoarticular arthritis was induced (FIG. 4). This wassignificant from day 4 onwards.

C57BL/6 mice treated with indomethacin showed significantly less paincompared with those mice not treated with indomethacin followingmBSA/IL-1 monoarticular arthritis induction (FIG. 4), such that thereadings were similar to GM-CSF−/− mice. As GM-CSF−/− did not exhibitpain, indomethacin treatment had no effect.

The significance levels are as follows:

-   -   C57BL/6 mice: indomethacin treated vs. untreated group (Group 4        vs. Group 3): day 4 p<0.0001; day 5, p<0.001; day 7, p=0.007    -   GM-CSF−/− mice vs. C57BL/6 mice (Group 1 vs. Group 3): day 4,        p<0.0001; day 5, p<0.0001; day 7, p=0.022

These results indicate that C57BL/6 mice develop significant pain fromday 4 onwards in a mBSA/IL-1 monoarticular arthritis model, whereasGM-CSF−/− mice do not show any significant signs of pain. Antagonists ofGM-CSF are therefore highly effective in the treatment of inflammatorypain.

Example 7 GM-CSF Antagonists are Effective in Treating InflammatoryPain/CFA Model

The following experiment is an additional experiment demonstrating theeffectiveness of GM-CSF antagonists in the treatment of inflammatorypain. Here, inflammatory pain was induced with Complete Freund'sAdjuvant. As in Experiment 5, pain was assessed with or withoutadministration of indomethacin, a pain relieving substance, at varioustime points using an incapacitance meter.

Mice

12 male C57BL/6 mice and 12 male GM-CSF−/− mice (see Example 1) wereused in each of the three treatment groups:

Group 1: C57BL/6 wildtype (n=12): CFA

Group 2: C57BL/6 wildtype (n=12): CFA+indomethacin

Group 3: GM-CSF KO (n=12): CFA

Induction of Inflammatory Pain

Complete Freund's Adjuvant (CFA) (Sigma) contains the heat-killedMycobacterium tuberculosis strain, H37Ra, in mineral oil at aconcentration of 1 mg/ml. CFA was mixed thoroughly by vortexing toensure that the heat-killed bacteria are incorporated in the suspension(Kamala T (Hock immunization: a humane alternative to mouse footpadinjections. J Immunol Methods 328:204-214.2007). Immediately aftervortexing, the adjuvant was drawn into a glass syringe using a 19-gaugeneedle. Bubbles were carefully eliminated from the syringe and theneedle was removed. Each mouse was injected subcutaneously in the lefthind paw (footpad) with 20 μl of the CFA emulsion. 1 mg/kg i.p.indomethacin (see Experiment 5) was administered to mice of Group 2, onehour before pain assessment.

Read Out for Pain

As in Experiment 5 an Incapacitance Tester (Dual Weight Averager) wasused for the automatic and reproducible assessment of analgesic potencyby measuring the weight distribution on the two hind paws. Weight placedon each hind limb was measured over a 5 second period. 3 separatemeasurements were taken per mouse for each time point then averaged.Results are expressed as injected limb/control limb×100. Thus a value of100 means that equal weight is being placed on the right and the leftlimb. A value below 100 means less weight is being placed on theinjected limb (left) compared with the control limb (right).Incapacitance was tested after 24, 48 and 72 h hours post injection ofCFA.

Results

Following s.c. injection of CFA into the left footpad, mice developedswelling of the left footpad, which was similar in magnitude in C57BL/6(Group 1) and GM-CSF−/− mice (Group 3). C57BL/6 mice treated withindomethacin (Group 2) also showed no difference in the degree ofswelling (see FIG. 5). There was no swelling of the contralateral(right) foot in any of the groups.

Assessment of weight distribution, as a measure of pain, showed thatC57BL/6 mice developed pain over time which was significantly greaterthan in GM-CSF−/− mice at 48 (p=0.03) and 72 (p=0.0009) hours post CFAinjection (FIG. 6). Strikingly GM-CSF−/− mice did not develop any pain.Treatment of C57BL/6 mice with indomethacin alleviated the pain suchthat the readings were no different to those for GM-CSF−/− mice (FIG.6). At 72 hrs post CFA injection C57BL/6 mice treated with indomethacinhad significantly less pain than C57BL/6 mice not treated withindomethacin (p=0.05).

Summary of Significance Levels:

48 hrs:

-   -   Group 3 vs. Group 1—p=0.03,    -   Group 2 vs. Group 1—p=0.09,

72 hrs:

-   -   Group 3 vs. Group 1—p=0.0009,    -   Group 2 vs. Group 1—p=0.05,

The degree of swelling of the footpad following CFA injection was nodifferent in GM-CSF−/− mice compared with C57BL/6 mice. Furthermore,indomethacin treatment of C57BL/6 mice had no effect on swelling, whichis likely due to the fact that it was only given one hour prior to theincapacitance readings. Thus the majority of swelling had alreadyoccurred before the first indomethacin injection was given at 24 hours.

In contrast, following CFA injection, C57BL/6 mice developed significantpain which was reduced by indomethacin. GM-CSF−/− mice, on the otherhand, did not show any signs of pain. Hence these experiments strikinglyshow that although the footpads of all mice are inflamed following CFAinjection, GM-CSF−/− mice do not show any signs of pain.

Example 8 Therapeutic Effectiveness of a GM-CSF Specific AntibodyComprising SEQ ID NOs. 1 or 2

Examples 2-7 are repeated, whereby as GM-CSF antagonist, a GM-CSFspecific antibody comprising an amino acid sequence of a heavy chainvariable region as depicted in SEQ ID No.:1 or comprising an amino acidsequence of a light chain variable region as depicted in SEQ ID No.:2 isused. Another species than mouse may be used, in particular a species towhich the antibody used in this experiment is cross reactive. Preferablythe animal species used in this experiment is rat.

The animals treated with the control antibody shows significantincreased signs of pain as compared to the animals which received aGM-CSF specific antibody comprising an amino acid sequence of a heavychain variable region as depicted in SEQ ID No.:1 or comprising an aminoacid sequence of a light chain variable region as depicted in SEQ IDNo.:2. This demonstrates the effectiveness of the antibodies in thetreatment of pain.

Example 9 Therapeutic Effectiveness of a GM-CSF Specific AntibodyComprising SEQ ID NOs. 3 or 4

Examples 2-7 are repeated. As GM-CSF antagonist, a GM-CSF specificantibody comprising an amino acid sequence of a heavy chain variableregion as depicted in SEQ ID NO:3 or comprising an amino acid sequenceof a light chain variable region as depicted in SEQ ID NO:4 is used.Another species than mouse may be used, in particular a species to whichthe antibody used in this experiment is cross reactive. Preferably theanimal species used in this experiment is rat.

The animals, e.g. rat, treated with the control antibody showsignificant increased signs of pain as compared to the animals whichreceived a GM-CSF specific antibody comprising an amino acid sequence ofa heavy chain variable region as depicted in SEQ ID NO:3 or comprisingan amino acid sequence of a light chain variable region as depicted inSEQ ID NO:4. This demonstrates the effectiveness of the antibodies inthe treatment of pain.

Example 10 Therapeutic Effectiveness of a GM-CSF Specific AntibodiesComprising SEQ ID NOs. 5-20

Examples 2-7 are repeated. As GM-CSF antagonist, a GM-CSF specificantibody comprising a H-CDR3 sequence selected from any one of SEQ IDNOs.5-16 is used. Preferably, said antibodies additionally comprise theH-CDR1 sequence of SEQ ID NO. 16, and/or the H-CDR2 sequence of SEQ IDNO.18, and/or the L-CDR1 sequence of SEQ ID NO:19, and/or the L-CDR2sequence of SEQ ID NO.20), and/or the L-CDR3 sequence of SEQ ID NO.21.Another species than mouse may be used, in particular a species to whichthe antibody used in this experiment is cross reactive. Preferably theanimal species used in this experiment is rat.

The animals, e.g. rat, treated with the control antibody showsignificant increased signs of pain as compared to the animals whichreceived a GM-CSF specific antibody according to the present example.This demonstrates the effectiveness of the antibodies in the treatmentof pain.

Example 11 Therapeutic Effectiveness of a GM-CSF Specific AntibodiesComprising SEQ ID NOs. 21-26

Examples 2-7 are repeated. As GM-CSF antagonist, a GM-CSF specificantibody comprising the L-CDR1 sequence of SEQ ID NO.22, and/or theL-CDR2 sequence of SEQ ID NO.23, and/or the L-CDR3 sequence of SEQ IDNO:24, and/or the H-CDR1 sequence of SEQ ID NO:25, and/or the H-CDR2sequence of SEQ ID NO:26, and/or the H-CDR3 sequence of SEQ ID NO:27 isused. Preferably said antibody comprise all the CRDs of SEQ IDNOs.22-28. Another species than mouse may be used, in particular aspecies to which the antibody used in this experiment is cross reactive.Preferably the animal species used in this experiment is rat.

The animals, e.g. rat, treated with the control antibody showsignificant increased signs of pain as compared to the animals whichreceived a GM-CSF specific antibody according to the present example.This demonstrates the effectiveness of the antibodies in the treatmentof pain.

Example 12 Therapeutic Effectiveness of Antibodies Specific for theGM-CSF Receptor

Examples 2-7 are repeated. As GM-CSF antagonist, a GM-CSF receptorspecific antibody comprising an amino acid sequence of a H-CDR3 sequencedepicted in any one of SEQ ID NO's.:28-46 is used. Another species thanmouse may be used, in particular a species to which the antibody used inthis experiment is cross reactive. Preferably the animal species used inthis experiment is rat.

The animals, e.g. rat, treated with the control antibody showsignificant increased signs of pain as compared to the animals whichreceived a GM-CSF receptor specific antibody according to the presentexample. This demonstrates the effectiveness of the antibodies in thetreatment of pain.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

BIBLIOGRAPHY

-   Brennan et al, Pain 64:493-501, 1996-   Ghose et al, J Combin Chem: 1:55-68, 1999-   Hargreaves et al, 1988-   Knappik et al, J. Mol. Biol. 296:57, 2000-   Krebs et al, J. Immunol. Methods. 254:67, 2001-   Kuzuna et al, Chem. Pharm. Bull. (Tokyo) 23:1184-1191, 1975-   Lipinski et al, Adv Drug Del Rev: 23:3-25, 1997-   Luger et al, Pain 99:397-406, 2002-   Pearson et al, Arthritis Rheum. 2:440-459, 1959-   Rothe et al, J. Mol. Biol. 376:1182, 2008-   Schwei et al, J: Neuroscience 19:10886-10897, 1999

The invention claimed is:
 1. A method for the treatment of pain in asubject, comprising administering to the subject an antagonist ofGM-CSF, wherein said antagonist is an antibody specific for GM-CSF orthe GM-CSF receptor, wherein said pain is selected from bone cancerpain, osteoarthritic pain and post-surgical pain.
 2. The method of claim1, wherein said subject is a human.
 3. The method of claim 1, whereinsaid antagonist is an antibody specific for GM-CSF.
 4. The method ofclaim 1, wherein said antagonist is an antibody specific for the GM-CSFreceptor.
 5. The method of claim 1, wherein said pain is bone cancerpain.
 6. The method of claim 1, wherein said pain is osteoarthriticpain.
 7. The method of claim 1, wherein said pain is post-surgical pain.